Anti-tumor drug, medicament, composition, and use thereof

ABSTRACT

The present invention relates to an active polypeptide including the amino acid sequence of SEQ ID NO:3, or having at least 50%, preferably 70%, more preferably 90% identity with the amino acid sequence of SEQ ID NO:3, or fragments thereof having at least 21 contiguous amino acids, or peptides having at least 50%, preferably 70%, more preferably 90% identity with the amino acid sequence of the fragments, provided that the polypeptide is not SEQ ID NO:2, variants and antigenic fragments thereof, SEQ ID NO 16 or SEQ ID NO 17, and uses thereof in a method of treating cancer and/or tumours of the human or animal body.

The present invention relates to the field of treatments for cancers.More specifically, the present invention relates to the treatment ofcancers by polypeptides derived from a protein belonging to thetetraspanin super family.

Cancer is a class of diseases or disorders characterized by uncontrolleddivision of cells and the ability of these cells to spread, either bydirect growth into adjacent tissue through invasion, or by implantationinto distant sites by metastasis (where cancer cells are transportedthrough the bloodstream or lymphatic system). Cancer may affect peopleat all ages, but risk tends to increase with age. It is one of theprincipal causes of death in developed countries.

There are many types of cancer. Severity of symptoms depends on the siteand character of the malignancy and whether there is metastasis. Oncediagnosed, cancer is usually treated with a combination of surgery,chemotherapy and radiotherapy. As research develops, treatments arebecoming more specific for the type of cancer pathology. Drugs thattarget specific cancers already exist for several cancers. If untreated,cancers may eventually cause illness and death, though this is notalways the case.

Current treatments target distinct properties of malignant cells, suchas for example evading apoptosis, unlimited growth potential(immortalization) due to overabundance of telomerase, self-sufficiencyof growth factors, insensitivity to anti-growth factors, increased celldivision rate, altered ability to differentiate, no ability for contactinhibition, ability to invade neighbouring tissues, ability to buildmetastases at distant sites, ability to promote blood vessel growth(angiogenesis).

Tumor angiogenesis is the proliferation of a network of blood vesselsthat penetrates into the tumor, supplying nutrients and oxygen andremoving waste products. Tumor angiogenesis actually starts withcancerous tumor cells releasing molecules that send signals tosurrounding normal host tissue. This signalling activates certain genesin the host tissue that, in turn, make proteins to encourage growth ofnew blood vessels. Solid tumors must stimulate the formation of newblood vessels in order to obtain the nutrients and oxygen necessary fortheir growth, thus providing a route by which the tumors can metastasizeto distant sites.

Experimental evidence has suggested that malignant tumors can induceangiogenesis through the elaboration of a variety of factors, such asacidic fibroblast growth factor (aFGF), basic fibroblast growth factor(bFGF), vascular endothelial growth factor (VEGF), platelet derivedgrowth factor (PDGF), transforming growth factor alpha (TGF-alpha),tumor necrosis growth factor alpha (TNF-alpha), and many others (Liottaet al., 1991, Cell 64: 327-336; Hanahan et al., Cell 86: 353-364).

Nowadays, plenty of chemotherapeutic molecules targeting angiogenesisare available on the market. Well known naturally occurring angiogenesisinhibitors are angiostatin, endostatin, interferons, platelet factor 4,prolactin 16 Kd fragment, thrombospondin, TIMP-1 (tissue inhibitor ofmetalloprotease-1), TIMP-2 and TIMP-3. These molecules can be used aschemotherapeutic treatments, as well as other drugs such as for examplecombrestatin A4, EMD 121974, TNP-470, squalamine, thalidomide,interferon-alpha, anti-VEGF, antibodies . . . . However, theirefficiency is never sufficient and alternative treatments are desirable.

There is therefore a need of alternative chemotherapeutic agents for thetreatment of tumors, having increased efficiency, being less invasive ortoxic, and resulting in an increased rate of recovery.

WO 03/074073, in the name of the Applicant, describes a family of 54genes involved in the regulation of angiogenesis. Amongst these genes,“gene 497” (SEQ ID No. 1 in this specification), which encodes “protein497” (SEQ ID No. 2 in this specification), also called TM4SF2 in WO03/074073, has been described as implied in the activation ofangiogenesis (pro-angiogenic). The expression of gene 497 is enhancedwhen angiogenesis is stimulated by pro-angiogenic factors such as VEGFand FGF2. WO 03/074073 also describes that the expression of an antisensof the gene 497, i.e. the inhibition of the expression of the gene 497,in human endothelial cells inhibits the formation of capillary tubes.

Bio-informatics analysis revealed that protein 497C, which comprises 244amino-acids, contains one extracellular loop SEL (Small ExtracellularLoop), one extracellular loop LEL (Large Extracellular Loop), fourtrans-membrane spans and two intracellular tails corresponding to the N-and C-terminals. Thus, this protein has been classified as a member ofthe tetraspanin super family (Levy et al., Nat Rev Immunol. 2005February; 5(2):136-48).

The tetraspanins are a large family of evolutionarily conservedcell-surface proteins that are expressed in a wide range of organisms.Members of this family tend to associate with each other, together withtheir partners, in membrane microdomains that provide a scaffold for thetransmission of external stimuli to intracellular-signalling components.Basically, tetraspanins comprise four transmembrane (TM) domains whichcontain conserved polar residues and flank the small and largeextracellular loops (SEL and LEL respectively). The LEL is composed of acore formed by helices a, b and e, and this core structure is conservedamong the tetraspanins. Helices c and d comprise the variable portion ofthe LEL, and they are flanked by the CCG motif and further conservedcysteine residues. This region is folded as a result of disulphidebridges to form a mushroom-like structure.

The members of this super-family have been classified into three groupsbased on the number of cysteine residues present in the LEL domain.Group 1 contains four cysteines in the LEL domain, group 2 contains sixcysteines in the LEL domain, and group 3 contains eight cysteines in theLEL domain.

Since protein 497C contains six cysteines within its LEL domain, itcould therefore be classified in group 2, similarly to protein CD151,another member of this family. The LEL domain of these proteinscomprises 6 domains: a, b, c, d1, d2 and e, among which d1, d2 and cconsist in the variable portions of the LEL.

Going deeper in their researches, the inventors produced truncated formsof protein 497C, corresponding to the various fragments of theextracellular domain SEL and LEL of protein 497C:

-   -   497C-T2: entire LEL domain of protein 497C, identified by SEQ ID        NO:3 in this specification (112 amino acids),    -   497C-T3: c, d1, d2 and e domains of the LEL domain, identified        by SEQ ID NO:4 in this specification (74 amino acids),    -   497C-T4: d1, d2 and e domains of the LEL domain, identified by        SEQ ID NO:5 in this specification (49 amino acids),    -   497C-T5: d2 and e domains of the LEL domain, identified by SEQ        ID NO:6 in this specification (43 amino acids).

To ensure the three-dimensional configuration of these fragments, i.e.the three-dimensional conformation of the LEL domain, and thereforetheir potential activity inventors added, according to an embodiment, atail at the C-terminus of the fragments. This tail was composed of arandom sequence of 30 to 70 amino acids, preferably of 45 to 65 aminoacids, more preferably of 50 to 60 amino acids, still more preferably ofabout 55 amino acids. This tail has no activity per se, and its presumedrole is to stabilize the three-dimensional structure of the LELfragment, i.e. to maintain the polypeptide folded in a biologicallyactive form.

In a first experiment, the inventors found that protein 497C-T2 mayinhibit human endothelial cell proliferation in vitro in a dosedependent manner.

Then, in a second experiment, the inventors surprisingly found that thetruncated forms of protein 497C may have a strong activity to inhibitcapillary tube formation in vitro, in a dose dependent manner.

Other experiments conducted by the inventors suggested that truncatedforms of protein 497C induced the inhibition of the migration ofendothelial cells in vitro, in a dose dependent manner. The results ofthe dose-response study on the inhibition of angiogenesis by 497C-T2revealed that at 270 nM, the recombinant protein 497C-T2 induced a smallinhibition while at 540 nM, 497C-T2 may inhibit by more than 50% invitro angiogenesis (i.e. IC₅₀<540 nM). This demonstrated that therecombinant protein 497C-T2 is a potent anti-angiogenic compound, andthat it could be at least 200-fold more potent than the anti-VEGF mAband/or VEGF receptor (KDR)-based identified peptides (Binetruy-TournaireR et al., Identification of a peptide blocking vascular endothelialgrowth factor (VEGF)-mediated angiogenesis, EMBO J. 2000; 19:1525-1533).

Capillary tube formation, human endothelial cell proliferation and humanendothelial cell migration are three essential steps of angiogenesis.Consequently, the fact that 497C-T2 may inhibit in vitro capillary tubeformation, human endothelial cell proliferation and/or migration in adose-dependant manner, thus constituted a strong evidence of the potentanti-angiogenic activity of the truncated forms of protein 497C.

All these results were absolutely unexpected since native protein 497Chas been disclosed as pro-angiogenic and that the expression of anantisens of the gene 497, i.e. the inhibition of gene 497, in humanendothelial cells inhibits the formation of capillary tubes. It wastherefore really surprising that truncated forms of protein 497C, mayhave anti-angiogenic activity.

In addition, inventors tested the effect of 497C-T2 on the proliferationof many cell lines, including the fibroblast cell line MRC5, CHO, thetumor cell lines Calu-6, NCI-H460, and more, without any detectableanti-proliferative effect even at high concentration. In contrast,497C-T2 significantly inhibited human endothelial cell proliferation forconcentration as low as 500 nM, reaching 75% inhibition at 5 μM. Hence,in addition of being more potent than the anti-VEGF therapeuticstrategies available today, 497C-T2 is highly specific to endothelialcells.

Interestingly, the inventors found that the truncated form 497C-T3,which lack the a and b domains, was almost as potent than 497C-T2 on theinhibition of angiogenesis and cell migration, indicating that the a andb domains only slightly contribute to the biological activity and/or tothe three dimensional conformation necessary for the biological activityof 497C-T2. The truncated forms 497C-T4 (lacking the a, b, and cdomains), and 497C-T5 (lacking the a, b, c, and d1 domains) had limitedactivities relative to that of 497C-T2, indicating that both the c andd1 domains are necessary for the activity of the protein 497C-T2.

These results were also unexpected, and led the inventors to test theanti-tumor activity of 497C-T2 in mice in vivo.

Still surprisingly, the inventors found that protein 497C-T2 had astrong anti-tumor activity in vivo, and a strong synergistic activity incombination with other chemotherapeutic agent such as for examplecisplatin.

Inventors found that the test substance 497C-T2 was not toxic in Nudemice bearing human NCI H460 or CALU-6 tumors at different tested doses.Moreover, 497C-T2 exhibited a strong statistically significantanti-tumoral activity against NCI H460 and CALU-6 tumors as early as twodays after the beginning of the treatment (2^(nd) IP injection combinedwith CDDP). This anti-tumoral activity was persistent during thetreatment period. The anti-tumoral effect of 497C-T2 in this model ofhuman lung cancer represented a realistic therapeutic approach as amonotherapy. Its efficacy was strongly potentiated when combined withthe cytotoxic anticancer drug CDDP (Cisplatin), which suppressed tumorgrowth. Cisplatin alone, on the other hand, did not eradicate tumorgrowth.

It is not clear whether the anti-angiogenic activity of 497C-T2 issolely responsible for its anti-tumoral activity. It is now acceptedthat biological compounds such as interferon, EGF and Her-2 receptorantagonists can modulate host responses and enhance the efficacy ofstandard chemotherapies. The data strongly suggested that 497C-T2 may beof use either as a primary anti-tumoral agent or as an add-on synergictherapy to primary cytotoxic agents for the treatment of cancers.

As mentioned above, it is now established that protein 497C is expressedin endothelial cells, and that its expression is enhanced whenangiogenesis is stimulated by pro-angiogenic factors such as VEGF andFGF2 (see WO 2003/074073). Protein 497C belongs to the Tetraspaninssuperfamily, which are transmembrane receptors comprising twoextracellular loops, implied in the recognition of extracellularsignals, and two intracellular tails (C-terminal and N-terminal),implied in the transduction of the signal. A key feature in the signaltransduction is the formation of a ligand-receptor complex between theextracellular loop of the receptor and the ligand. Without wanting to bebound with a theory, Applicants think that the truncated forms of 497Cmay play their role through a “soluble receptor mechanism”: truncatedforms of 497C may remain soluble on the surface of the cell and may berecognized by the ligand. As a result, there may be a competition in therecognition of the ligand between the soluble forms of 497C (thefragments of the invention) and the native transmembrane protein, andconsequently a decrease, in a dose dependent manner, of the transductionof the pro-angiogenic signal, therefore resulting in the inhibition ofangiogenesis and then in the decrease of tumour volume.

The present invention thus relates, in a first aspect, to an activepolypeptide comprising the amino acid sequence of SEQ ID NO:3, or havingat least 50%, preferably 70%, more preferably 90% identity with theamino acid sequence of SEQ ID NO:3, or fragments thereof having at least21 contiguous amino acids, or peptides having at least 50%, preferably70%, more preferably 90% identity with the amino acid sequence of saidfragments, provided that said polypeptide is not SEQ ID NO:2, antigenicfragments thereof, SEQ ID NO 16 or fragments thereof and SEQ ID NO 17 orfragments thereof.

As used herein, “peptide” means short molecules formed from the linking,in a defined order, of less than 100 amino acids.

As used herein, “polypeptide” means molecules formed from the linking,in a defined order, of at least 100 amino acids.

As used herein, “active polypeptide” means polypeptides which have abiological activity. In the present invention the polypeptides have ananti angiogenic and anti tumour activity.

In an embodiment of the invention, said active polypeptide comprises theamino acid sequence of SEQ ID NO:3, or has at least 50%, preferably 70%,more preferably 90% identity with the amino acid sequence of SEQ IDNO:3, or comprises fragments thereof haying at least 21 contiguous aminoacids, or peptides having at least 50%, preferably 70%, more preferably90% identity with the amino acid sequence of said fragments, providedthat said polypeptide is not SEQ ID NO:2, variants and antigenicfragments thereof, SEQ ID NO 16 or fragments thereof and SEQ ID NO 17 orfragments thereof.

A “variant” of SEQ ID NO 2 refers to SEQ ID NO 2 wherein one, two ormore amino acids are mutated. Such variants include, for example,deletions from, or insertions or substitutions of, residues within SEQID NO 2 or any combination of deletion, insertion and substitution. Oneexample of variants of SEQ ID NO 2 is SEQ ID NO 37 corresponding to the249 mer transmembrane 4 superfamily member 2 protein (P41732) whichsequence has five additional N-terminal amino acids compared to SEQ IDNO 2.

In one embodiment of the invention, said active polypeptide comprisesthe amino acid sequence of SEQ ID NO:3, or has at least 50%, preferably70%, more preferably 90% identity with the amino acid sequence of SEQ IDNO:3, or comprises fragments thereof having at least 21 contiguous aminoacids, or peptides having at least 50%, preferably 70%, more preferably90% identity with the amino acid sequence of said fragments, providedthat said polypeptide is not SEQ ID NO:2 and antigenic fragmentsthereof, variants of SEQ ID NO 2 such as SEQ ID NO 37, SEQ ID NO 16 orfragments thereof and SEQ ID NO 17 or fragments thereof.

In an embodiment, the polypeptides according to the invention furthercomprise means for having it folded in an active three dimensionalconformation. Preferably, said means for having it folded in an activeconformation consists in any sequence comprising from 30 to 70 aminoacids, preferably from 45 to 65 amino acids, more preferably from 50 to60 amino acids, still more preferably of about 55 amino acids, saidsequence being fused to the C-terminus of said polypeptide. In apreferred embodiment, said sequence being fused to the C-terminus ofsaid polypeptide is SEQ ID NO 18 (DRASP QPWRYRIRIL APSTSLRPHS STTTTTTXIRLLTKPERKLS WLLPPLSNN).

As used herein, “three dimensional conformation” means the tertiarystructure of a polypeptide, i.e. its overall shape, in which thepolypeptide performs a biological function.

As used herein, the term “fragments” means truncated forms of the LELdomain of protein 497C, having an anti-tumor activity. Said fragmentspreferably have an amino acid sequence of at least 21 contiguous aminoacids of SEQ ID NO:3. In a particular embodiment, the fragments have anamino acid sequence of at least 43 contiguous amino acids. In anotherparticular embodiment, said fragments have the amino acid sequence ofSEQ ID N:4 (74 amino acids), SEQ ID N:5 (49 amino acids) or SEQ ID N:6(43 amino acids). Fragments also include peptides having at least 50%,preferably 70%, more preferably 90% identity with the amino acidsequence of said fragments, and having an anti-tumor activity.

In another embodiment, said fragments have the following sequences:

SEQ ID NO 19: DE RSRAVDHVQRSLSCCGVQ NYTNWSTSPY FLEHGIPPSCCMNETDCNPQ DLHNLTVAAT KVNQKGCYDL VTSFMETNMG IIAG SEQ ID NO 20:AAT KVNQKGCYDL VTSFMETNMG IIAG SEQ ID NO 21:ISGFVFRHEI KDTFLRTYTD AMQTYNGNDE RS SEQ ID NO 22:YNGNDE RSRAVDHVQR SLSCCGVQN SEQ ID NO 23:SLSCCGVQNY TNWSTSPYFL EHGIPPSCCM N SEQ ID NO 24: EHGIPPSCCM NETDCNPQDLHSEQ ID NO 25: ETDCNPQDL HNLTVAATKV NQKG SEQ ID NO 26:ISGFVFRHEI KDTFLRTYTD AMQTYNGNDE RSRAVDHVQR SLSCCGVQNY SEQ ID NO 27:ISGFVFRHEI KDTFLRTYTD AMQTYNGNDE RSRAVDHVQRSLSCCGVQNY TNWSTSPYFL EHGIPPSCCM N SEQ ID NO 28:ISGFVFRHEI KDTFLRTYTD AMQTYNGNDE RSRAVDHVQRSLSCCGVQNY TNWSTSPYFL EHGIPPSCCM NETDCNPQDL SEQ ID NO 29:ISGFVFRHEI KDTFLRTYTD AMQTYNGNDE RSRAVDHVQRSLSCCGVQNY TNWSTSPYFL EHGIPPSCCM NETDCNPQDL HNLTVAATKV NQKGSEQ ID NO 30: YNGNDE RSRAVDHVQR SLSCCGVQNY TNWSTSPYFL EHGIPPSCCM NSEQ ID NO 31: YNGNDE RSRAVDHVQR SLSCCGVQNY TNWSTSPYFLEHGIPPSCCM NETDCNPQDL SEQ ID NO 32:YNGNDE RSRAVDHVQR SLSCCGVQNY TNWSTSPYFLEHGIPPSCCM NETDCNPQDL HNLTVAATKV NQKG SEQ ID NO 33:YNGNDE RSRAVDHVQR SLSCCGVQNY TNWSTSPYFLEHGIPPSCCM NETDCNPQDL HNLTVAATKV NQKGCYDLVT SFMETNMGII AG SEQ ID NO 34:SLSCCGVQNY TNWSTSPYFL EHGIPPSCCM NETDCNPQD SEQ ID NO 35:SLSCCGVQNY TNWSTSPYFL EHGIPPSCCM NETDCNPQDL HNLTVAATKV SEQ ID NO 36:IPPSCCM NETDCNPQDL HNLTVAATKV NQKGPeptides having at least 50%, preferably 70%, more preferably 90%identity with the amino acid sequence of said fragments, and having ananti-tumor activity are also included in the invention.

In an embodiment, said fragments of SEQ ID NO 3 having at least 21contiguous amino acids are selected among SEQ ID NO 4 to 6 and SEQ ID NO19 to 36, or any peptides having at least 50%, preferably 70%, morepreferably 90% identity with said fragments.

In a preferred embodiment, said fragments of SEQ ID NO 3 having at least21 contiguous amino acids are selected among SEQ ID NO 4, SEQ ID NO 5and SEQ ID NO 6 or peptides having at least 50%, preferably 70%, morepreferably 90% identity with said fragments.

In a second aspect, the present invention relates to a medicamentcomprising a polypeptide, a fragment, and/or a peptide as describedabove.

In a third aspect, the present invention relates to a pharmaceuticalcomposition comprising a polypeptide, a fragment, and/or a peptide asdescribed above, and one or more pharmaceutically-acceptable excipients.

In a fourth aspect, the invention relates to a pharmaceuticalcomposition comprising a polypeptide, a fragment, and/or a peptide asdescribed above, and one or more pharmaceutically-acceptable excipients,for use in a method of treatment of cancer and/or tumors of the human oranimal body.

In a particular embodiment, the pharmaceutical compositions as describedabove further comprise at least one other active substance selected fromanti-angiogenic substances or anti-tumor substances. These substancesmay be chosen by the man in the art, regarding the effect to beachieved. Preferably, these substances can be selected from cisplatin,carboplatin, etoposide, ifosfamide, mitomycin, vinblastine, vinorelbine,gemcitabine, paclitaxel, docetaxel, and irinotecan, etc.

In a fifth aspect, the invention relates to a pharmaceutical compositioncomprising synergistic effective amounts of

-   -   a polypeptide, a fragment, and/or a peptide as described above,        and    -   a platinum complex selected from the group consisting of        cisplatin and carboplatin.

The medicament or composition useful in the practice of this inventionis administered to the mammal by known conventional routes. Themedicament or composition described herein may be administered by thesame route, or by different routes. For example, the medicament orcomposition may be administered to patients orally or parenterally(intravenously, subcutaneously, intramuscularly, intraspinally,intraperitoneally, and the like).

When administered parenterally, the composition is preferably formulatedin a unit dosage injectable form (solution, suspension, emulsion) withat least one pharmaceutically acceptable excipient. Such excipients aretypically nontoxic and non-therapeutic. Examples of such excipients arewater, aqueous vehicles such as saline, Ringer's solution, dextrosesolution, and Hank's solution and non-aqueous vehicles such as fixedoils (e.g., corn, cottonseed, peanut and sesame), ethyl oleate, andisopropyl myristate. Sterile saline is a preferred excipient. Theexcipient may contain minor amounts of additives such as substances thatenhance solubility, isotonicity, and chemical stability, e.g.,antioxidants, buffers, and preservatives. When administered orally (orrectally) the compounds will usually be formulated into a unit dosageform such as a table, capsule, suppository, or cachet. Such formulationstypically include a solid, semi-solid or liquid carrier or diluent.Exemplary diluents and excipients are lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineraloil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin,methylcellulose, polyoxyethylene, sorbitan monolaurate, methylhydroxybenzoate, propyl hydroxybenzoate, talc and magnesium stearate. Inpreferred embodiments, the pharmaceutical composition according to theinvention is administered intravenously.

According to the invention, the amount of polypeptide present in themedicament or composition is effective to treat susceptible tumors.Preferably, the polypeptide is present in an amount from 0.01 to 90% inweight, preferably from 0.1% to 10% in weight, more preferably from 1%to 5% in weight, in the medicament or in the composition. These amountsare routinely adaptable by the man in the art, who is able to choose thebest quantity to administer to a patient to achieve recovery.

In a sixth aspect, the invention relates to the use of the polypeptide,fragment, and/or of the peptide as described above, or of the medicamentas described above, or of the pharmaceutical composition as describedabove, for the treatment of cancers and/or tumors.

According to the invention, the tumors to be treated are preferablysolid tumors. More preferably, the tumors to be treated are selectedfrom sarcomas, carcinomas, and lymphomas. Examples of such tumors arebladder cancer, melanoma, breast cancer, non-Hodgkin's lymphoma, braincancer, bone cancer, colon and rectal cancer, liver cancer, pancreaticcancer, endometrial cancer, prostate cancer, kidney cancer, skin cancer(non-melanoma), thyroid cancer, lung cancer (small cell lung cancer andnon small cell lung cancer).

In a seventh aspect, the present invention relates to a method oftreatment comprising administering to a subject in need of treatment thepolypeptide, fragment, and/or the peptide as described above, or themedicament as described above, or the pharmaceutical composition asdescribed above, in an amount sufficient to inhibit cancer or tumorgrowth.

In a particular embodiment, the invention relates to the method oftreatment as described above further comprising administering at leastone other anti-neoplastic or anti-tumor drug.

In these methods, administering comprises topical administration, oraladministration, intravenous administration, or intraperitonealadministration.

In an eight aspect, the present invention relates to a method oftreatment comprising administering to a subject in need of treatment asynergistic effective amount of

-   -   a polypeptide, a fragment, and/or a peptide as described above,        and    -   a platinum complex selected from the group consisting of        cisplatin and carboplatin, which is sufficient to inhibit cancer        or tumor growth.

In one embodiment, said polypeptide or fragments thereof and saidplatinum complex are administered simultaneously.

In another embodiment, said polypeptide or fragments thereof and saidplatinum complex are administered sequentially. Preferably, saidpolypeptide or fragments thereof and said platinum complex areadministered by separate routes, i.e. orally or parenterally(intravenously, subcutaneously, intramuscularly, intraspinally,intraperitoneally, and the like).

In a particular embodiment, said platinum complex is cisplatin.

In another particular embodiment said platinum complex is carboplatin.

The present invention will now be further described with reference tothe following non-limiting examples.

FIG. 1 is a diagram representing the % inhibition of endothelial cellproliferation in vitro with increasing concentrations of protein497C-T2.

FIGS. 2 a, 2 b, 2 c, 2 d, 2 f, 2 g, 2 h and 2 i are pictures of in vitroangiogenesis of endothelial cells in different conditions:

-   -   FIG. 2 a: Control (Buffer Urea 2M)    -   FIG. 2 b: 497C-T2 6.5 μg/mL (0.27 μM)    -   FIG. 2 c: 497C-T2 13 μg/mL (0.54 μM)    -   FIG. 2 d: 497C-T2 26 μg/mL (1.08 μM)    -   FIG. 2 e: 497C-T2 48 μg/mL (2 μM)    -   FIG. 2 f: 497C-T3 51 μg/mL (2.55 μM)    -   FIG. 2 g: 497C-T4 55 μg/mL (3.18 μM)    -   FIG. 2 h: 497C-T5 56 μg/mL (3.35 μM)    -   FIG. 2 i: C-terminal tail 53 μg/mL (4.14 μM)

FIGS. 3 a, 3 b, 3 c, 3 d, 3 e, 3 f, 3 g are pictures of wound assay onendothelial cells performed in different conditions:

-   -   FIG. 3 b: 497C-T2 20 μg/mL (0.83 μM)    -   FIG. 3 c: 497C-T2 40 μg/mL (1.66 μM)    -   FIG. 3 d: 497C-T3 48 μg/mL (2.4 μM)    -   FIG. 3 e: 497C-T4 60 μg/mL (3.52 μM)    -   FIG. 3 f: 497C-T5 61 μg/mL (3.65 μM)    -   FIG. 3 g: C-terminal tail 63 μg/mL (4.92 μM)

FIG. 4 is a graph representing Mean Tumor Volume (mm³) versus time(days) for different groups of mice treated according to example 7.

FIG. 5 is a graph representing Mean Relative Tumor Volume (without unit)versus time (days) for different groups of mice treated according toexample 7.

FIG. 6 is a graph representing Mean Tumor Volume (mm³) versus time(days) for different groups of mice treated according to example 8.

FIG. 7 is a graph representing Mean Relative Tumor Volume (without unit)versus time (days) for different groups of mice treated according toexample 8.

EXAMPLE 1 Production of Protein 497C-T2

Synthesis of Insert 497C-T2:

First, gene 497C was cloned in pGEM®-T easy vector system (Promega®)according to known procedures (the vector obtained was called“pGEM-T-497C”).

Second, the insert T2 (SEQ ID NO:3) corresponding to the extra-cellularpart LEL of the protein 497C was amplified by PCR using the plasmid“pGEM-T-497C” and the two primers CDS5 (SEQ ID NO:9) and CDS4 (SEQ IDNO:10) (table 1), which frame the LEL domain in 5′ and 3′, respectively.

TABLE 1 Primer SEQ ID NO Sequence 497c-cds-5 9GACGACGACAAGATttcagggtttgtgtttcgtcatgagatcaa 497c-cds-4 10GAGGAGAAGCCCGGT ctccagcgatgattcccatgtt

Third, the DNA sequence coding for the protein 497C-T2 was inserted intothe vector pET-30 EK/LIC (Novagen®) according to known procedures(pET-30-497C-T2). The nucleic acid sequence coding for 497C-T2 withinthe pET-30 vector is given in SEQ ID NO:7.

The purified vector was then introduced in E. coli BL21(DE3) pLys forprotein production. Colonies were controlled for the presence of boththe vector end the insert by PCR.

The size of the produced protein 497C-T2 was 24 kD, and it was higherthan the expected size. This was due to the addition of a supplementarytail at the C-terminal (54 random amino acids) as well as of a His-Tagat the N-terminal as confirmed by sequencing. The amino acid sequence ofthe protein 497C-T2 as produced is given in SEQ ID NO:8. The protein497C-T2 was produced within the insoluble fraction of the bacteria,which necessitated an extraction in denaturating conditions.

Extraction and Purification of the Protein 497C-T2

Following culture, bacteria were lyzed, centrifuged and the supernatantdiscarded. The insoluble fraction obtained was treated with Tris-HCl 20mM, urea 8 M, imidazol 5 mM, NaCl 0.5 M, GSH 5 mM, pH 8.0. After thistreatment, the suspension was centrifuged and the supernatant collected,filtered on 0.45 μm membranes to discard insoluble materials. Thefiltered extract was then used to purify the protein 497C-T2 by using aHis-Trap column (Amersham®) connected to a HPLC system (Amersham).

The purified protein obtained was diluted in 4 M urea and 0.3 Mimidazol. To remove these agents from the preparation, the solution wassubjected to dialysis at 4° C.

Following these steps of dialysis, the purified protein was centrifugedat 4,000×g for 15 min and filtered on 0.45 μm membranes to eliminatepossible precipitates. The purified protein preparation was controlledfor protein content according to the method described by Bradford in1976 (Anal. Biochem. 72:248-54) and by SDS-PAGE. The gels were analyzedusing the Gene Genius software to quantify the purity by image analysis.

To increase purity of the protein 497C-T2, we performed a secondpurification step by using ion exchange liquid chromatography. TheHisTrap purified preparation was diluted 3 times with the bufferTris-HCl 20 mM, pH 8, 2 M urea (to decrease the concentration of NaCl to50 mM), and loaded on MonoS column connected to a HPLC system run byUnicorn software (Amersham, GE, Saclay, France). The column was thenwashed extensively and eluted with a linear gradient of ionic force(0.05 M to 0.5 M NaCl in the Tris-HCl 20 mM buffer, pH 8, 2 M urea). Thepurified protein preparation was controlled for protein content both byBradford and by SDS-PAGE.

EXAMPLE 2 Design and Production of the Truncated Forms 497C-T3, 497C-T4,and 497C-T5

To identify the active site of the protein 497C-T2, three otherstruncated forms called 497C-T3, 497C-T4, and 497C-T5 were designed andproduced as well as a control polypeptide corresponding to both theN-terminal attached His-Tag and the C-terminal tail coming from thevector. To do so, 5′ specific primers were designed allowing theprogressive truncation of the N-terminal sequence of the protein 497C-T2without affecting the N-terminal His-Tag necessary for the purificationof the produced truncated forms.

497C-T3

The truncated 497C-T3 was built by PCR amplification of the DNA usingthe vector pET30-497C-T2 as template, the 5′ specific primer 497c-cds7(SEQ ID NO:11), which permitted to eliminate most of the b domain and topreserve the first C-C bridge, and the 3′ specific primer 497C-cds4 (SEQID NO:10).

TABLE 2 Primer SEQ ID NO Sequence 497c-cds7 115′GACGACGACAAGATGCAGCGCAGCCTGAGCTGC 3′ 497c-cds-4 10 3 ′GAGGAGAAGCCCGGTctccagcgatgattcccatgtt 5′497C-T4

The truncated 497C-T4 was built by PCR amplification of the DNA usingthe vector pET30-497C-T2 as template, the 5′ specific primer 497c-cds9(SEQ ID NO:12), which permitted to eliminate the first C-C bridge, the band c domains, and to preserve the d1 domain, and the 3′ specific primer497C-cds4 (SEQ ID NO:10).

TABLE 3 Primer SEQ ID NO Sequence 497c-cds9 12 5′GACGACGACAAGATCCCCCCCAGCTGCTGCATG 3′ 497c-cds-4 10 3 ′GAGGAGAAGCCCGGTctccagcgatgattcccatgtt 5′497C-T5

The truncated 497C-T4 was built by PCR amplification of the DNA usingthe vector pET30-497C-T2 as template, the 5′ specific primer 497c-cds11(SEQ ID NO:13), which permitted to eliminate the first and the secondC-C bridges, the b, c and d1 domains, and to preserve the linearsequence corresponding to the d2 and e domains, and the 3′ specificprimer 497C-cds4 (SEQ ID NO:10).

TABLE 4 Primer SEQ ID NO Sequence 497c-cds11 13 5′GACGACGACAAGATGAACGAAACTGATTGTAATCCCC 3′ 497c-cds-4 10 3 ′GAGGAGAAGCCCGGTctccagcgatgattcccatgtt 5′

For the N-terminal His-Tag and the C-terminal tail, the constructs wereobtained using the following primers which totally eliminated thesequence of the protein 497C-T2.

TABLE 5 Primer SEQ ID NO Sequence Pet30 cdsl 145′GACGACGACAAGATGGACCGGGCTTCTCCT 3′ pet30 cds2 155′GAGGAGAAGCCCGGTCTAGTTATTGCTCAGCGG 3′The amplified products corresponding to the truncated forms 497C-T3,497C-T4, 497C-T5, and the C-terminal tail were cloned and produced asdescribed for the protein 497C-T2.

EXAMPLE 3 Test of Inhibition of Endothelial Cell Proliferation by 497-T2In Vitro

Cell Culture

HUVEC cells were cultured to confluency in complete EGM2-MV medium(Cambrex) at 37° C. and in 5% CO₂ humidified atmosphere. Cells were thencollected by trypsine-EDTA digestion (Versene, Eurobio). After 5 min,the enzymatic reaction was stopped by adding 3 ml of the culture mediumcontaining 5% FCS. Cells were then centrifuged at 220 g for 10 min atroom temperature, washed twice with 5 ml of culture medium, suspended incomplete culture medium, counted and adjusted to 50 000 cells/ml. Onehundred μL per well were then distributed to a 96-well cell culturegrade micro-plate (5 000 cells/well) and incubated with differentconcentrations of the purified protein 497C-T2 in Tris-HCl 20 mM buffer(pH 8), containing 150 mM NaCl and urea 2M; this buffer was used ascontrol.

After 42 hrs at 37° C., cell proliferation was measured using thiazolylblue tetrazolium bromide (MTT) method. Briefly, MTT (Sigma) wasdissolved in PBS at 5 mg/ml, the solution was filtered (0.22 μm) and 10μl were added to each well of the 96-well micro-plates. After 3 hrs ofincubation at 37° C., 5% CO₂ humidified atmosphere, the micro-plateswere centrifuged at 220×g for 10 min, the supernatant was discarded, andthe crystals dissolved by the addition of 100 μl of DMSO to each well.The optical density (OD) at 570 nm was then measured using μQuantmicro-plate reader (Bio-Tek Instrument gmbh, Colmar, France) coupled tothe KC4 (Bio-Tek) software. The OD was corrected by subtractingblank-well OD values (the OD values obtained from wells without cells),and the inhibition of cell proliferation was measured relative tocontrol (OD obtained from wells with untreated HUVEC representing themaximal proliferative response, i.e. 100%).

As shown in FIG. 1, protein 497C-T2 inhibited human endothelial cellproliferation in a dose dependent manner. This inhibition represented77% at 122 μg (i.e. 5 μM) of protein 497C-T2.

EXAMPLE 4 Inhibition of In Vitro Angiogenesis by 497-T2, 497C-T3,497C-T4 and 497C-T5

The purified proteins 497C-T2, 497C-T3, 497C-T4 and 497C-T5 were testedin vitro on angiogenesis of HUVEC induced by FGF2 and VEGF on Matrigel.

24 wells plates were prepared with 250 μL of BD Matrigel™/well and thenincubated 30 minutes in incubator. HUVEC cells were then prepared asdescribed in example 3 and 70 000 cells were seeded per well andincubated with different concentrations of the purified protein 497C-T2,497C-T3, 497C-T4 or 497C-T5 in Tris-HCl 20 mM buffer (pH 8), containing150 mM NaCl and urea 2M; this buffer was used as control:

-   -   FIG. 2 a: Control (Buffer Urea 2M)    -   FIG. 2 b: 497C-T2 6.5 μg/mL (0.27 μM)    -   FIG. 2 c: 497C-T2 13 μg/mL (0.54 μM)    -   FIG. 2 d: 497C-T2 26 μg/mL (1.08 μM)    -   FIG. 2 e: 497C-T2 48 μg/mL (2 μM)    -   FIG. 2 f: 497C-T3 51 μg/mL (2.55 μM)    -   FIG. 2 g: 497C-T4 55 μg/mL (3.18 μM)    -   FIG. 2 h: 497C-T5 56 μg/mL (3.35 μM)    -   FIG. 2 i: C-terminal tail 53 μg/mL (4.14 μM)

As shown in FIGS. 2 b, 2 c, 2 d and 2 e, protein 497C-T2 inhibited invitro angiogenesis in a dose-dependent manner.

Moreover, as shown in FIGS. 2 f, 2 g and 2 h, the truncated forms497C-T3, 497C-T4 and 497C-T5 showed different levels of activity:497C-T3 (FIG. 2 f) and 497C-T4 (FIG. 2 g) were almost as efficient as497C-T2 for the anti-angiogenic activity, suggesting that the a, b and cLEL domains are not necessary for the anti-angiogenic activity ofprotein 497C-T2. To the contrary, 497C-T5 (FIG. 2 h) showed only aresidual inhibitory activity, while the C-terminal tail was totallyinactive (FIG. 2 i).

EXAMPLE 5 Inhibition of the Migration of Human Endothelial Cells by497-T2, 497C-T3, 497C-T4 and 497C-T5

Cell migration was tested by the wound assay described by Sato andRifkin (J Cell Biol. 1988; 107:1199) with few modifications. HUVEC grownin growth medium EGM-2MV (Cambrex) were seeded in 24-well plates at 80000 cells per well in 500 μL of growth medium and grown to confluence at37° C. in a humidified atmosphere containing 5% CO₂. Cells were scrappedwith a plastic tip on one line only. After wounding, the culture mediumwas changed for fresh medium (control, FIG. 3 a) or fresh mediumsupplemented with:

-   -   FIG. 3 b: 497C-T2 20 μg/mL (0.83 μM)    -   FIG. 3 c: 497C-T2 40 μg/mL (1.66 μM)    -   FIG. 3 d: 497C-T3 48 μg/mL (2.4 μM)    -   FIG. 3 e: 497C-T4 60 μg/mL (3.52 μM)    -   FIG. 3 f: 497C-T5 61 μg/mL (3.65 μM)    -   FIG. 3 g: C-terminal tail 63 μg/mL (4.92 μM)

After 18 hours of culture, cells were observed and photographed underthe inverted microscope (Analysis, Olympus, Rungis, France).

As shown in FIGS. 3 b and 3 c, protein 497C-T2 inhibited humanendothelial cells migration in a dose dependent manner.

Moreover, as shown in FIGS. 3 d, 3 d, 3 e and 3 f the truncated forms497C-T3, 497C-T4 and 497C-T5 showed different levels of activity. Thetruncated form 497C-T3 inhibited cell migration similarly to the protein497C-T2 (FIG. 3 d). To the contrary, the truncated form 497C-T4 (FIG. 3e) was less active than 497C-T2, 497C-T5 (FIG. 3 f) showed very limitedanti-migratory activity, and the C-terminal tail (FIG. 3 g) was totallyinactive. These results thus suggested that the a and b LEL domains arenot necessary for the anti-migratory activity of protein 497C-T2.

EXAMPLE 6 Expression of Protein 497C in Tumor Samples

A number of different human tumor samples were screened for theexpression of the gene 497C. For each pathological sample, the peripheryof the tumor was separated from the core of the tumor, and theexpression of the gene 497C in these two area was compared after mRNAextraction followed by RT-PCR.

Kidney Tumor Samples

19 pathological biopsies from kidney tumors were analysed. In 13 out of19 patients, the expression of 497C was much higher in the core than inthe periphery of the tumor.

Lung Tumor Samples

40 pathological biopsies from human lung tumors were analysed. In 37 outof 40 patients, the expression of 497C was much higher in the peripherythan in the core of the tumor. There was also a close relationshipbetween the level of expression of 497C at the periphery of the tumorand the patient's nod status (i.e. the metastasis potential). Theanatomical examination of the samples also revealed that the peripheryof the tumor was much more vascularised than the core of the tumor (asestablished for lung cancer in general).

Colon Tumor Samples

33 pathological biopsies from human colon tumors were analysed. In 25out of 33 patients, the expression of 497C was much higher in theperiphery than in the core of the tumor.

EXAMPLE 7 Test of 497C-T2 on NCI H460 Human Tumor in Swiss Nude Mice InVivo

Preparation of NCI H460 Cells

NCI H460 cells were cultured as adherent cells in complete RPMI 1640medium (Ref. CM1RRM08-01, batch No. 623615, Eurobio, France) containing2 mM L-Glutamine, adjusted to 4.5 g/L glucose (Ref. G7528, batch No.033K0121, Sigma, France) and supplemented with 10 mM HEPES (Ref. H0887,batch No. 113K2338, Sigma, France), 1.0 mM sodium pyruvate (Ref.CSTVAT00-0U, batch No. 520818, Sigma, France) and 10% fetal calf serum(FCS; Ref. CVFSVF00-01, batch No. S13021, Eurobio, France) under a 37°C., 5% CO₂ humidified atmosphere. They were amplified in 75 cm²-flasksto reach 90×10⁶ cells.

At D0, NCI H460 cells (human lung carcinoma) were collected from 75cm²-flasks by removing the medium and adding 3 ml of trypsine-EDTA (Ref.CEZTDA00-0U, batch No. 633920, Eurobio, France). After 5 min ofincubation at 37° C., cells were detached from the plastic and theenzymatic reaction was stopped by adding 3 ml of RPMI 1640 mediumcontaining 10% fetal calf serum. Cells were then centrifuged at 700 gfor 5 min at room temperature. They were resuspended in serum-free RPMI1640 culture medium containing L-Glutamine (2 mM), glucose (4.5 g/l),sodium pyruvate (1 mM) and buffered with HEPES (10 mM). Cells werecounted and the number of viable NCI H460 cells was >99%. The number ofcells was then adjusted to 25.10⁶ cells/ml in serum-free medium.

Tumor Induction

Thirty healthy female Swiss Nude mice were anesthetized by IP injectionof Ketamine-Xylazine (80 mg/kg-12 mg/kg; Ref. K-113, Sigma, France). NCIH460 cells (5.10⁶ cells/mouse in 200 μL of serum-free medium) were thenimplanted subcutaneously in the right flank of each mouse.

Treatment Schedule

At D12 post-implantation of the NCI H460 cells, the thirty mice wererandomized into six groups of 5 mice. Tumor volumes had reached 228 to468 mm³ and mean tumor volumes were not statistically different betweengroups after randomization.

The treatment schedule, starting D12 and ending D28, is summarized inTable 6:

-   -   Animals of group 1 were treated with the vehicle solution (Batch        C): Tris-HCl pH 7.5, 2M Urea, 150 mM NaCl, 0.1 mM CaCl₂,    -   Animals of group 2 were treated with a solution of cisplatin in        physiological serum 0.5 ml/mL (CDDP, cis-diamineplatinum (II)        dichloride, Ref. P4394, batch No. 014K0993, Sigma, France,        purity 100%, MW. 300),    -   Animals of groups 3, and 4 were treated with the vehicle        solution supplemented with 1 mg/mL of the test substance 497C-T2        (Batch A),    -   Animals of group 5 were treated with the vehicle solution        supplemented with 1 mg/mL of the test substance 497C-T2 (Batch        A), and further received CDDP.    -   Animals of group 6 were treated with the vehicle solution        supplemented with 1.8 mg/mL of the test substance 497C-T2 (Batch        B), and further received CDDP.

All solutions were injected IP. Injections in groups 1, 2, 3, 5 and 6were performed according to the schedules Q2DX8, i.e. 1 quantity everytwo days, eight times. Injections in group 4 were performed according tothe schedules Q1DX16, i.e. 1 quantity every day, sixteen times.

CDDP was resuspended in sterile physiological serum at a concentrationof 0.5 mg/mL and injected IP at a concentration of 5 mg/kg at a volumeof 10 mL/kg, according to the treatment schedule Q2DX8.

Mice were observed for 2 h post-injection. Ketamine/Xylazine (80mg/kg-12 mg/kg; Ref. K-113, Sigma, France) was used to anaesthetize theanimals before sacrifice by cervical dislocation.

Monitoring of the Mice

Animals have been observed daily. Modification in animal behavior hasbeen reported in the laboratory notebook. Body weights and tumor volumeswere recorded every two days until the end of the experiment.

Data outlined below were calculated:

-   -   Tumor growth curves were drawn using the mean tumor volumes        (MTV),    -   Mean Relative tumor volume (MRTV) was calculated as the ratio        between the MTV at time t and the volume at the time of        injection (t=D12),    -   Tumor growth inhibition (T/C, %) was evaluated as the ratio of        the median tumor volumes of treated groups versus vehicle group.

TABLE 6 Animals Administration Treatment dose Administration TreatmentGroup n Treatment route (mg/kg/adm) volume schedule 1 5 Vehicle (BatchC) IP 0 10 ml/kg Q2DX8 2 5 Cisplatin IP 5 Q2DX8 3 5 497C-T2 (Batch A) IP10 Q2DX8 4 5 497C-T2 (Batch A) IP 10 Q1DX16 5 5 497C-T2 (Batch A) & IP10 Q2DX8 Cisplatin 5 Q2DX8 6 5 497C-T2 (Batch B) & IP 18 Q2DX8 Cisplatin5 Q2DX8

TABLE 7 Mean body weight (MBW) of mice bearing NCI H-460 tumors treatedwith the vehicle, CDDP at 5 mg/kg (schedule Q2DX8, G2), 497C-T2 at 10.0mg/kg (schedule Q2DX8, G3), 497C-T2 at 10.0 mg/kg (schedule Q1DX16, G4),combined 497C-T2 at 10.0 mg/kg and CDDP at 5 mg/kg (schedule Q2DX8, G5),and combined 497C-T2 at 18.0 mg/kg and CDDP at 5 mg/kg (schedule Q2DX8,G6) at D12 and D28. Treatment dose MBW at D12 MBW at D28 MBWC D12-D28Group Test substance (mg/kg) (g) (g) (g) 1 Vehicle-Batch C 0 22.53 ±0.79 23.72 ± 0.72  1.18 ± 0.79 2 Cisplatin 5.00 21.29 ± 0.41 18.74 ±1.59 −2.55 ± 1.83 3 497C-T2-Batch A 10.0 24.06 ± 1.59 25.70 ± 1.12  1.63± 1.59 4 497C-T2-Batch A 10.0 21.76 ± 1.63 23.68 ± 2.17  1.91 ± 1.36 5497C-T2-Batch A + 10.0 23.28 ± 2.03 20.19 ± 0.66 −3.09 ± 2.68 Cisplatin5.00 6 497C-T2-Batch B + 18.0 23.08 ± 1.78 19.00 ± 2.00 −4.08 ± 0.71Cisplatin 5.00Statistical Studies

Statistical analyses of tumor volumes (V), time to reach ‘V’,tumor-doubling time (DT), relative tumor volume (RTV) and tumor growthinhibition (T/C) were performed for all groups. Data are expressed asmean±SD. Groups of data were normally distributed. Univariate analysiswere performed to assess differences between groups. Statisticalsignificance was then determined using the Student's t test. A P<0.05was considered as statistically significant. The Statistical analysiswas performed using XLSTAT (Addinsoft, France).

Body Weight Monitoring

As shown in table 7, the vehicle had no impact on the body weight: mousebehavior and body weight gain were normal and no animal diedprematurely. No toxicity was observed during the course of the treatmentwith the test substance 497C-T2 at both doses tested (10 and 18 mg/kg).In contrast, an important toxicity was observed in groups 2, 5 and 6treated with CDDP (from −12 to −18% body weight loss; p<0.05).

The results of mean tumor volume (MTV), mean relative tumor volume(MRTV), tumor volume and tumor growth parameters are shown in FIGS. 4, 5and in Table 8 and Table 9.

As shown in table 8, the MTV was decreased at D28 in mice of group 2treated with CDDP (1440±1097 mm³) compared to mice of the vehicle group1 (4441±1135 mm³). The MTV at D28 was also decreased in groups 3 and 4treated with the test substance 497C-T2 at 10 mg/kg with 1 injection perday (2482±2075 mm³) and 1 injection every other day (3167±1681 mm³),respectively. A massive reduction of the MTV was observed in animalsfrom group 5 (807±692 mm³) and group 6 (639±416 mm³) compared to group1, the vehicle treated animals (4441±1135 mm³).

The T/C ratio, which is a parameter of tumor growth inhibition,represented 32% in mice from group 2 at D28 demonstrating that CDDPreduces by 68% tumor size compared to the vehicle-treated group 1. T/Cwas 56% in mice from for group 3 and 71% in mice from group 4, revealinga moderate anti-tumoral activity of the test substance when used as amonotherapy. When combined with CDDP however, T/C fell to 18% and 14% inmice from groups 5 and 6, respectively. This demonstrates that 497C-T2has a potent anti-tumoral activity when combined a cytotoxic agent suchas CDDP.

The tumor size doubling time (DT) was 1.23±0.49 days in thevehicle-treated group 1. The DT increased 4-fold in mice of group 2treated with CDDP alone (5.12±2.89 days). For groups 3 and 4, the DT(4.08±1.4 and 5.9±1.21 days, respectively) was similar to that of group2. This indicates that 497C-T2 is as potent as CDDP to reduce tumoractivity when used as monotherapy. But most importantly, bi-therapyusing 497C-T2 and CDDP increased 20-fold DT in animals from groups 5 and6 (25.02±29.89 and 20.07±9.28 days, respectively) compared to the DTmeasured in mice from the vehicle-treated group 1. This further confirmsthe potent anti-tumoral activity of the tested substance 497C-T2 whenused alone or in combination with CDDP.

As shown in table 9, MRTV measures confirmed that animal of group 6presented the strongest decrease of the tumor volume, at bothconcentration tested (group 5, 6). Treatment with 497C-T2 alone led to aMRTV at D28 of 7.37 (group 3) or 6.76 (group 4) and treatment withcisplatin led to a MRTV of 4.88 (group 2).

All these results confirm that 497C-T2 is a potent anti-tumor agent,either used alone or in a synergistic combination with cisplatin.

EXAMPLE 8 Test of 497C-T2 on Non-Small Human Cell Lung Cancer (CALU-6)Xenograft Model in Swiss Nude Mice In Vivo

Preparation of CALU-6 Cells

CALU-6 cells were cultured as adherent cells in complete EMEM medium(Ref. CM1MEM18-01, batch No. 462502, Eurobio, France) 10% fetal calfserum (FCS; Ref. CVFSVF00-01, batch No. 513021, Eurobio, France) under a37° C., 5% CO₂ humidified atmosphere. They were amplified in 75cm²-flasks to reach 90×10⁶ cells.

At D0, CALU-6 cells (human lung carcinoma) were collected from 75cm²-flasks by removing the medium and adding 3 ml of trypsine-EDTA (Ref.CEZTDA00-0U, batch No. 633920, Eurobio, France). After 5 min ofincubation at 37° C., cells had detached from the plastic and theenzymatic reaction was stopped by adding 3 ml of EMEM medium containing10% fetal calf serum. Cells were then centrifuged at 700 g for 5 min atroom temperature. They were resuspended in serum-free EMEM culturemedium. Cells were counted and viability assessment by Trypan Blueexclusion (Ref. CSTCOL03-OU, batch No. 434511, Eurobio, France). Thenumber of viable CALU-6 cells was >99%. The number of cells was thenadjusted to 25×10⁶ cells/ml in serum-free medium.

TABLE 8 Monitoring of human NCI H460 tumor growth in Nude mice after IPtreatment with vehicle, 497C-T2 (10 mg/kg and 18 mg/kg) and CDDP (5mg/kg). Treatment MTV at MTV at T/C at T/C at T/C at dose MTV at D14 D24D28 D14 D24 D28 DT Group Test substance (mg/kg) (mm³) (mm³) (mm³) (%)(%) (%) (days) 1 Vehicle-Batch C 0 999.64 ± 3222.36 ± 4440.68 ± 100 100100 1.23 ± 443.46 494.51 1135.16 0.49 2 CDDP 5.0 513.35 ± 1083.74 ±1440.18 ± 51.53 33.63 32.43 5.12 ± 333.36 899.44 1097.29 ^(a) 2.89 3497C-T2-Batch A- 10.0 571.00 ± 1955.02 ± 2481.61 ± 57.12 60.67 55.884.08 ± 306.73 1687.55 2075.18 1.4 4 497C-T2-Batch A- 10.0 468.85 ±2289.22 ± 3167.34 ± 46.90 71.04 71.32 5.9 ± 250.00 1170.70 1680.99 1.215 497C-T2-Batch A- + 10.0 388.79 ± 671.93 ± 807.43 ± 38.89 20.85 18.1825.02 ± CDDP 5.0 315.14 570.47 ^(a) 692.22 ^(a, c) 29.89 6 497C-T2-BatchB- + 18.0 459.45 ± 561.71 ± 638.74 ± 45.96 17.43 14.38 20.07 ± CDDP 5.0166.55 ^(a) 218.34 ^(a, b, c, d) 415.53 ^(a, b, c, d) 9.28 MTV: meantumor volume; T/C: treated/vehicle tumor volume ratio; DT: doubling timeof tumor volume. ^(a) p < 0.05 vs G1 ^(b) p < 0.05 vs G2 ^(c) p < 0.05vs G3 ^(d) p < 0.05 vs G4.

TABLE 9 Mean relative tumor volume (MRTV) of animals bearing the NCIH460 tumor and treated with vehicle (Group 1), CDDP alone (Group 2),497C-T2 administered every other day (Group  3) or once daily (Group 4),and CDDP combined with 497C-T2 at the lower (Group 5) and higher dose(Group 6). D12 D14 D16 D18 D20 D22 D24 D26 D28 Group 1 1.00 3.18 4.396.87 9.28 11.61 9.73 14.15 19.50 Group 2 1.00 1.59 1.74 2.30 2.29 3.393.67 3.67 4.88 Group 3 1.00 1.37 1.70 2.46 2.81 3.30 3.22 5.80 7.37Group 4 1.00 1.08 1.00 1.69 2.63 3.08 3.08 4.89 6.76 Group 5 1.00 1.011.18 1.34 1.75 2.18 1.80 2.04 2.45 Group 6 1.00 1.24 1.06 0.89 1.07 1.181.17 1.30 1.48Tumor Induction

Thirty healthy female Swiss Nude mice were anesthetized by IP injectionof Ketamine-Xylazine (80 mg/kg-12 mg/kg; Ref. K-113, Sigma, France).CALU-6 cells 5×10⁶ cells/mouse in 200 μl of serum-free medium) were thenimplanted subcutaneously in the right flank of each mouse. Mice wereobserved for 2 h post-implantation.

Treatment Schedule

At D12 post-implantation of the CALU-6 cells, the thirty mice wererandomized into six groups of 5 mice. Tumor volumes had reached 228 to468 mm³ and mean tumor volumes were not statistically different betweengroups after randomization.

The treatment schedule, starting D12 and ending D28, is summarized inTable 10.

-   -   Animals of group 1 were treated with the vehicle solution        (Tris-HCl pH 7.5, 2M Urea, 150 mM NaCl, 0.1 mM CaCl₂) (Batch C);    -   Animals of group 2 were treated with a solution of cisplatin in        physiological serum 0.5 mh/mL (CDDP, cis-diamineplatinum(II)        dichloride, Ref. P4394, batch No. 014K0993, Sigma, France,        purity 100%, MW. 300),    -   Animals of group 3 were treated with the vehicle supplemented        with the test substance 497C-T2 at a dose of 10 mg/kg.    -   Animals of group 4 were treated with the vehicle supplemented        with the test substance 497C-T2 at a dose of 10 mg/kg, and        further received CDDP.

Injections in groups 1, 2, 3 and 4 were performed according to theschedules Q2DX8, i.e. 1 quantity every two days, eight times.

CDDP was resuspended in sterile physiological serum at a concentrationof 0.5 mg/mL and injected IP at a concentration of 5 mg/kg at a volumeof 10 mL/kg, according to the treatment schedule Q2DX8.

Mice were observed for 2 hours post-injection. Ketamine/Xylazine (80mg/kg-12 mg/kg; Ref. K-113, Sigma, France) was used to anaesthetize theanimals before sacrifice by cervical dislocation. For all animals, thetumor size was measured twice a week with calipers. The tumor volume(mm³) was measured according to the formula: (length×width²)/2 (4).

Statistical Studies

Mean tumor volumes (MTV), mean relative tumor volume (MRTV) and tumorgrowth inhibition (T/C) were calculated as for example 7.

Body Weight

As shown in table 11, the vehicle had no impact: mouse behavior and bodyweight gain were normal and no animal died prematurely (excepted mouse 5of group 1). No toxicity was observed during the course of the treatmentwith the test substance 497C-T2 at the dose of 10 mg/kg, a slight bodyweight gain was observed (+1.44 g).

In contrast, an important toxicity was observed in groups 2, 4 treatedwith CDDP. (12.6% and 8.7% body weight loss respectively). Thedifference between group 1 versus 2 and 4 and group 3 versus 2 and 4 wasstatistically significant (p<0.0001) but the difference between group 2and 4 was not statistically significant.

TABLE 10 Animals Administration Treatment dose Administration TreatmentGroup n Treatment route (mg/kg/adm) volume schedule 1 5 Vehicle IP 0 10ml/kg Q2DX8 2 5 Cisplatin IP 5 Q2DX8 3 5 497C-T2 IP 10 Q2DX8 4 5 497C-T2& IP 10 Q2DX8 Ciplatine 5 Q2DX8

TABLE 11 Mean body weight (MBW) of mice bearing CALU-6 tumors treatedwith the vehicle, CDDP at 5 mg/kg (schedule Q2DX8, G2), 497C-T2 at 10.0mg/kg (schedule Q2DX8, G3), combined 497C-T2 at 10.0 mg/kg and CDDP at 5mg/kg (schedule Q2DX8, G4) at D12 and D28. Treatment dose MBW at D12 MBWat D28 MBWC D12-D28 Group Test substance (mg/kg) (g) (g) (g) 1 Vehicle 022.35 ± 1.17 24.40 ± 1.14 +2.04 2 Cisplatin 5.00 21.39 ± 0.85 18.69 ±1.97 −2.70 3 497C-T2 10.0 21.67 ± 1.32 23.11 ± 1.97 +1.44 4 497C-T2 +Cisplatin 10.0&5.00 21.94 ± 0.74 19.84 ± 1.89 −2.11

The results of mean tumor volume (MTV), mean relative tumor volume(MRTV), tumor volume and tumor growth parameters are shown in FIGS. 6, 7and in Table 12 and Table 13.

The MTV was decreased at D28 in mice of group 2 treated with CDDP(742.44±215.85 mm³) compared to mice of the vehicle group 1 (1233.44±663,82 mm³). The MTV at D28 was also decreased in group 3 treatedwith the test substance 497C-T2 at 10 mg/kg with 1 injection per twodays (813.70±439,00 mm³). These results were confirmed by the analysisof the MRTV at D28 (table 13). A massive reduction of the MTV wasobserved in animals from group 4 (430.89±290.89 mm³) compared to group1, the vehicle treated animals (1 233.44±663,82 mm³).

The difference between group 1 and the 2 groups treated with the testsubstance reach the statistical significativity (p<0.0001 vs 4−p=0.003vs 3). The difference between group 2 and 4 was also significant(p=0.001). In contrast no statistical difference was observed betweengroup 2 (CDDP alone) and group 3 (497c-T2 alone). The first statisticalsignificativity between group 1 and treated group was observedrespectively at D22 for groups 3 and D20 for group 4. These results wereconfirmed by the analysis of the MRTV at D28 (table 13). An importantreduction of the MTV was observed in animals from group 4 (430.89±290,89mm³) compared to group 1, the vehicle treated animals (1 233.44±663,82mm³).

TABLE 12 Growth inhibition based on T/C ratio T/C ratio (%) Day D14 D16D18 D20 D22 D24 D26 D28 G2 −70%  24% −6%  17% 11% 15% 45% 27% G3 10% 33%7% 13% 26% 24% 39% 27% G4 22% 39% 8% 28% 30% 41% 60% 64%

The T/C ratio (table 12), which is a parameter of tumor growthinhibition, reveals a slight anti-tumoral activity of the test substancewhen used as a monotherapy as it reduces by 27% tumor size compared tothe vehicle-treated group 1. However, when combined with CDDP, theinhibition rate reach 64% reduction of tumor size relative to thevehicle-treated group 1. These results directly demonstrate that 497C-T2has a potent anti-tumoral activity when it was used in combination witha cytotoxic agent such as CDDP.

TABLE 13 Mean Relative tumor volume (MRTV) of animals bearing NCI H-460cells and treated with vehicle (group 1), CDDP alone (Group 2), 497C-T2(10.0 mg/kg) (Group 3), or combined with 497C-T2 and CDDP (Group 4)according to the scheduled treatment Q2DX8. MRTV Group D12 D14 D16 D18D20 D22 D24 D26 D28 1 1 1.83 3.17 2.51 3.68 4.16 5.68 7.49 8.24 2 1 3.112.39 2.66 3.08 3.70 4.84 4.11 5.98 3 1 1.64 2.11 2.34 3.22 3.09 4.314.60 6.02 4 1 1.42 1.92 2.32 2.66 2.89 3.33 2.96 2.96

As shown in table 13, MRTV reached 2.96 at D28 for the animals of group4, which confirm the synergistic efficacy of 497C-T2 with Cisplatin.Moreover, cisplatin used alone (group 2) or 497C-T2 used alone (group 3)showed very close MRTV at D28, suggesting that 497C-T2 is also a potentmono-therapy anti-tumor agent.

1. An active amino acid sequence comprising at least one of SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, provided that said aminoacid sequence is not: (i) SEQ ID NO:2, (ii) variants of SEQ ID NO: 2selected from the group consisting of SEQ ID NO:2 wherein 1 to 5 aminoacids are mutated and SEQ ID NO: 37, (iii) SEQ ID NO 16 or (iv) SEQ IDNO
 17. 2. The amino acid sequence according to claim 1, furthercomprising means for having it folded in an active three dimensionalconformation.
 3. The amino acid sequence according to claim 2, whereinsaid means for having it folded in an active conformation consists ofany sequence comprising from 30 to 70 amino acids being fused to theC-terminus of said amino acid sequence.
 4. The amino acid sequenceaccording to claim 1, having the amino acid sequence of SEQ ID NO: 4,SEQ ID NO: 5 or SEQ ID NO:
 6. 5. The amino acid sequence according toclaim 1, having an anti-tumour activity.
 6. A medicament comprising anamino acid sequence according to claim
 1. 7. A pharmaceuticalcomposition comprising an amino acid sequence according to claim 1, andone or more pharmaceutically-acceptable excipients.
 8. A pharmaceuticalcomposition comprising an amino acid sequence according to claim 1, andone or more pharmaceutically-acceptable excipients, for use in a methodof treatment of cancer and/or tumours of a human or animal body.
 9. Apharmaceutical composition according to claim 7, further comprising atleast one other active substance selected from anti-angiogenicsubstances or anti-tumour substances.
 10. A pharmaceutical compositioncomprising synergistically effective amounts of an amino acid sequenceaccording to claim 1, and a platinum complex selected from the groupconsisting of cisplatin and carboplatin.
 11. The pharmaceuticalcomposition according to claim 7, being in a form suitable for topical,systemic, oral, subcutaneous, transdermal, intramuscular orintra-peritoneal administration.
 12. The pharmaceutical compositionaccording to claim 7, wherein said amino acid sequence is present in anamount from 0.01 to 90% by weight.
 13. A method of treating cancersand/or tumours, comprising: administering an effective amount of theamino acid sequence according to claim 1 to a subject in need thereof.14. The method according to claim 13, wherein the tumours are solidtumours.
 15. The method according to claim 14, wherein the solid tumoursare selected from sarcomas, carcinomas, and lymphomas.
 16. A method forinhibiting cancer and/or tumour growth comprising administering to asubject in need of treatment the amino acid sequence according to claim1, in an amount sufficient to inhibit cancer or tumour growth.
 17. Themethod of claim 16, wherein said tumour is a solid tumour.
 18. Themethod of claim 16, further comprising administering at least one otherantineoplastic or anti-tumor drug.
 19. The method according to claim 16,wherein administering comprises topical administration, oraladministration, intravenous administration, or intraperitonealadministration.
 20. A method for inhibiting cancer or tumour growthcomprising administering to a subject in need of treatment asynergistically effective amount of an amino acid sequence according toclaim 1, and a platinum complex selected from the group consisting ofcisplatin and carboplatin, which is sufficient to inhibit cancer ortumour growth.
 21. The method of claim 20, wherein said amino acidsequence and said platinum complex are administered simultaneously. 22.The method of claim 20, wherein said amino acid sequence and saidplatinum complex are administered sequentially.
 23. The method of claim20, wherein said amino acid sequence and said platinum complex areadministered by separate routes.
 24. The method according to claim 20,wherein said platinum complex is cisplatin.
 25. The method according toclaim 20, wherein said platinum complex is carboplatin.